Radiotherapy is utilized by 80% patients as a part of cancer treatment (Nair et al., 2001). Ionizing radiation (IR) causes generation of reactive oxygen species (ROS) and has deleterious effects on cells (Kalpana et al., 2011). Radiation attenuates the endogenous antioxidant enzymes which maintain redox balance and normal biochemical processes (Prabhakar et al., 2007). The two complementary strategies with drugs to enhance therapeutic index of radiotherapy are to increase radiation-induced cell death in tumor and reduce damage in surrounding normal tissues. This can be achieved by modulation of DNA repair, cell cycle, signal transduction pathway, normal tissue damage and/or increase in radio-sensitization of tumor (Begg et al., 2011). Radioprotectors protect normal cell from radiation induced damage. Mechanisms for radioprotection includes inhibition of free radicals generation or acceleration of scavenging free radicals, enhancement of DNA and membrane repair, reconstruction of HP function and stimulation of immune cell activity (Wang et al., 2013). Amifostine (WR2721) is a clinically approved radioprotector in cancer treatment for reducing side effects in patients undergoing radiotherapy (Brizel et al., 2000). Other known radioprotectors are methylproamine, PrC-210 and ON01210/Ex-RAD® (Kamran et al., 2016). These radioprotectors are associated with limitation by route of administration and related toxicity. CBLB502 had shown an excellent selective radioprotection to healthy cells over cancerous cells through constitutive activation of NFκB pathway as Toll-Like receptor 5 agonist (Burdelya et al., 2008).
The DNA ligands such as bisbenzimidazoles Hoechst 33342 and Hoechst 33258, form strong and non-covalent linkages, with the adenine and thymine rich regions in the minor groove of DNA, significantly altering the chromatin structure. Administration of these compounds prior to irradiation afford protection against the formation of primary lesions in the aqueous solutions of DNA as well as in the intact cell nucleus. These DNA ligands have also been observed to reduce the radiation induced cytogenetic damage and cell death in cell cultures, as well as in whole body irradiated animals (Singh et al., 1998; Young and Hill, 1989). However, post-irradiation treatment of cells with these ligands has been observed to enhance cell death in vitro (Singh et al., 1998). Free radical scavenging and quenching of DNA radicals appear to be the mechanisms responsible for protection by Hoechst compounds administered prior to irradiation, but its role in enhancing the radiation-induced cell death when administered after irradiation is not clearly understood.
The limitations of these minor groove binding ligands as being mutagenic, clatogenic and cytotoxic because of the DNA lesions caused on account of topoisomerase I inhibition, gene expression alteration and repair inhibition prevent them from being used in humans. Therefore, the development of DNA binding ligands (Minor Groove Binding Ligands particularly) that afford radioprotective effect without significant mutagenicity and cytotoxic effects can play a significant role in biological radiation protection. Although numbers of radioprotectors are developed, there is only one approved radioprotector. Therefore there is necessity for alternative, nontoxic and effective radioprotector with multiple modes of actions for better radioprotection.
Our earlier in vitro work proved DMA as non-toxic free radical scavenging radioprotector (Kaur et al., 2012; Singh and Tandon, 2011; Tawar et al., 2007; Tawar et al., 2003). It did not show toxicity in vivo at maximum tolerated dose (MTD) of 2000 mg/kg. DMA was effective to deliver radioprotective effect at 1/7 dose of its MTD at 8Gy TBI (Nimesh et al., 2015). DMA induces NIK mediated NFκB activation and modulates number of key regulatory pathways including effector proteins (TP53, HSP70, SET, NPM and UBC) to overcome radiation induce damage (Kaur et al., 2012; Ranjan et al., 2013). In the present invention investigation has been carried out for the molecular mechanism to decipher the ability of DMA to protect normal and tumor bearing Balb/c mice against radiation-induced HP/GI injury, regulation of cellular antioxidant level, and modulatory effect on mRNA expression. The inventors showed that single 200 mg/kg oral and 50 mg/kg intravenous (i.v.) DMA dose augments 80 and 100% survival at 8Gy respectively through Akt/NFκB pathway, maintenance of antioxidant enzymes, improving HP & GI conditions and modulation of genes in TBI in vivo.